The agent of plague, Yersinia pestis, is a facultative intracellular pathogen that can survival and replicate in macrophages. Although the intracellular replication phase of Y. pestis is likely to play a key role in pathogenesis, especially during the early stages of plague, very little is known about this aspect of Y. pestis virulence. To elucidate the mechanism of Y. pestis survival and replication in macrophages, the following specific aims are proposed. First, genetic approaches will be used to identify Y. pestis genes that are required for intracellular proliferation in macrophages (rip genes). The proteins encoded by rip genes will be studied using molecular, cellular and immunological techniques to reveal the underlying basis for Y. pestis proliferation in macrophages. Second, DNA microarray analysis will be performed to characterize the global gene expression profile of Y. pestis replicating inside or outside of macrophages. These experiments will identify genes that are differentially expressed in the two environments. With this information in hand we will have a better understanding of how Y. pestis adapts its physiology to survive and replicate in macrophages. Third, mice will be experimentally infected by the aerosol route with wild-type Y. pestis or with rip mutants generated in aim 1. Bacterial growth in lungs, liver and spleen will be measured as a function of time to determine how intracellular proliferation contributes to virulence. Cell labeling and microscopic techniques will be used to detect intracellular bacteria in tissue sections and to identify host cell types that harbor intracellular bacteria. These combined approaches will lead to a better understanding of Y. pestis pathogenicity, and will help foster the development of new strategies to prevent or treat plague in the human population.